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Method for preparing promoter of Brassica napus BnPABP2 and application thereof

A promoter, pbi-pbnabp2 technology, applied in the field of plant genetic engineering and biology, can solve the problems of harmful ecological environment, unsuitable production practice, and unfavorable normal plant growth.

Inactive Publication Date: 2012-08-29
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of inducible promoters also has certain limitations. The external condition treatment of recipient plants, such as heat shock, hormone treatment, etc., may cause a series of physiological and biochemical reactions in the organism, which is not conducive to the normal growth of plants.
Moreover, the methyl dehydrocortisol (dex, dexamethasone), estradiol (estradiol) and tetracycline (tetracycline) used as inducers in the chemical regulation system are all harmful to the ecological environment and should not be used in production practice.

Method used

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  • Method for preparing promoter of Brassica napus BnPABP2 and application thereof
  • Method for preparing promoter of Brassica napus BnPABP2 and application thereof
  • Method for preparing promoter of Brassica napus BnPABP2 and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Rapeseed P BnPABP2 Expression pattern analysis of driven endogenous genes:

[0057] The rapeseed used in the present invention is Brassica napus L. . The test materials were sown in the field and managed normally.

[0058] Take roots, stems, leaves, flower buds, and siliques from fertile plants with the same growth conditions in the field. Take at least three replicates of each material, and at least one plant for each replicate. After sampling, wrap them in tin foil and place them in liquid nitrogen immediately. medium, stored at -80°C. RNA was extracted (method described above). The fluorescent quantitative PCR instrument is RTTM-Cycler (Boao Biological Co., Ltd.), using Rapeseed Actin (accession number AF111812) as the internal standard gene, Actin gene primers are forward primer 5'-CTGGAATTGCTGACCGTATGAG-3' and reverse primer 5'- ATCTGTTGGAAAGTGCTGAGGG-3'. Rapeseed P BnPABP2 The driven endogenous gene primers were 5'-GGCTTCAAATCTGGCTAATG-3' and 5'-TGGAGAACTTCG...

Embodiment 2

[0063] A brassica napus promoter P BnPABP2 The preparation method of promoter, its step is:

[0064] A. Prepare rapeseed P BnPABP2 Promoter primer design:

[0065] Utilize the SDS cracking method to extract the rapeseed DNA (J. Sambrook.D.W.Russell, Huang Peitang et al. Translation Molecular Cloning Test Guide (Third Edition) Science Press), according to the genome walking kit (purchased from Clontech company, product Codenamed Cat.No.638904) operating procedures, genome walking, walking for two rounds of PCR amplification, cloned to obtain rapeseed P BnPABP2 (942bp). The primers used for walking are listed in Table 2.

[0066] Table 2 Genome walking clone P BnPABP2 Primers used

[0067]

[0068] B. Brassica napus promoter P BnPABP2 The preparation is:

[0069] According to the instructions of the genome walking kit (purchased from Clontech, product code Cat. For genomic DNA, four "libraries" of restriction-enzyme-treated DNA ligated with specific adapters were est...

Embodiment 3

[0073] pBI-P BnPABP2 Arabidopsis transformation and PCR detection:

[0074]Arabidopsis thaliana was transformed according to the transformation method in the above literature (Zhang X R. et al. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature, 2006, 1:1-6). According to the unique kanamycin resistance of the transgenic plants, the green shoots obtained were preliminarily considered to be positive shoots when they were grown on MS (described above) medium containing 50 mg / L kanamycin. After the green seedling grows two true leaves, it is transplanted into vermiculite, and after the inflorescence appears on the plant, a true leaf is taken to extract genomic DNA (described above) by SDS method for PCR identification. The primer sequences are 5′-GAATTGTGAGCGGATAAC-3′ and 5′-ACATAAGGGACTGACCAC-3′; the PCR reaction system is as follows: Genomic DNA template 1μL (about 50ng), 10×Taq enzyme reaction buffer 2ul, 25mMMgCL 2 1.2ul, 2mM ...

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Abstract

The invention discloses a method for preparing a promoter (PBnPABP2) of Brassica napus BnPABP2 and the application thereof. The operation of cloning a PBnPABP2 sequence by using a genome walking method comprises the following steps: extracting genome DNA by using a SDS cracking process; in different systems, carrying out restriction endonuclease reaction on the extracted DNA respectively by usingendonucleases DraI, EcoRV, PvuII and StuI; after the obtained DNA is connected with a linker fragment, carrying out first-round PCR amplification by taking the linker fragment as a template; and carrying out second-round PCR amplification by taking a50-time diluent of the obtained first-round PCR product as a template, thereby obtaining the PBnPABP2. The PBnPABP2 is applied in the roots, stems, leaves, flower buds, anthers and peels of plants. The promoter has the function of driving the expression of exogenous genes, and the expression sites thereof are respectively in the roots, stems, leaves, flower buds, anthers and peels of plants; and the promoter has application potentials in the aspect of safety of edible oil of Brassica napus and the aspects of improving the disease resistance, stress resistance and lodging resistance of crops, improving the quality of crops, artificially creating a male sterile line and a restoring line, enriching germ plasm resources, and the like through transgenosis.

Description

technical field [0001] The invention relates to the fields of plant genetic engineering and biotechnology. Specifically related to a Brassica napus BnPABP2 promoter (named P BnPABP2 , the same below), and also relates to a preparation method of Brassica napus BnPABP2 promoter. The present invention also relates to the carrier containing the promoter or its homologous nucleotide sequence and the application of the promoter in genetic engineering of rapeseed and other plants. Background technique [0002] Plant promoters play a key role in the regulation of gene expression. The regulation of gene expression is the result of the comprehensive action of many factors. Generally, according to the sequence of events, gene regulation is divided into transcription level regulation, translation level regulation, and protein processing level regulation. The proteins and RNAs encoded by genes and their secondary metabolites are extremely important for maintaining the whole life acti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/84A01H5/06A01H5/04A01H5/12A01H5/02A01H5/08
Inventor 刘胜毅石磊董彩华黄军艳刘越英
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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